Abstract Over 40 genetic mutations have been identified in patients with nephrotic syndrome over the last 20 years. These mutations often alter structural (such as actin) and functional (such as GTPase) molecules important for podocytes. The goal of this proposal was to study downstream factors that may be regulated by these genes. Our central hypothesis is that loss of a transcription factor serum response factor(SRF) leads to loss of profilin 1(PFN1) expression causing altered podocyte actin stress fiber formation and RhoA gtpase activity. Our hypothesis is supported by data generated by the applicant that: 1) bioinformatic analysis of knockout mice in the mentor's laboratory and genes that when mutated cause nephrotic syndrome in humans have an altered SRF pathway; 2) Loss of podocyte specific Srf expression(SrfKO) leads to the development of albuminuria; 3) SrfKO podocytes have reduced stress fiber formation, reduced spreading ability, and reduced migration; 4) Enrichment of SRF ChIP-seq data suggests the importance of Pfn1 expression in podocytes; 5) SrfKO podocytes have reduced Pfn1 expression; 6) Podocyte specific Pfn1 knockout(Pfn1KO) mice develop albuminuria; 7) Pfn1KO podocytes have reduced spreading ability and reduced migration. The rationale for the proposed work is that better understanding of the SRF pathway will enable new strategies to prevent and treat nephrotic syndrome. We will test our hypothesis and address critically important questions in two specific aims. In our first specific aim, we will phenotype podocyte specific Pfn1 knockout mice and use translating ribosome affinity purification(TRAP) technology to isolate podocyte specific RNA to identify an early molecular profile of podocyte injury in vivo. In our second specific aim, we will perform in vitro studies to determine the molecular mechanisms by with SrfKO and Pfn1KO lead to altered stress fiber formation and stationary phenotypes. These results will have a positive impact by providing new targets that may improve our ability to treat nephrotic syndrome patients.